DK153555B - METHOD OF ANALOGY FOR THE PREPARATION OF 20-AMINO DERIVATIVES OF TYLOSIN, DESMYCOSIN, MACROSIN OR LACTENOCIN OR ACID ADDITION SALTS. - Google Patents

Description

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The present invention relates to an analogous process for the preparation of a novel group of 20-amino derivatives of tylosin, desmycosin, macrocine or lactenocin or acid addition salts thereof. These derivatives 5 are valuable as antibiotics.

Improved antibiotics are needed continuously. In addition to antibiotics useful in treating human diseases, improved antibiotics are also needed in the veterinary field. Increased potency, expanded spectrum 10 for bacterial inhibition, increased in vivo efficacy and improved pharmaceutical properties such as greater oral absorption, higher blood or tissue concentrations, longer body half-life and more advantageous degree or mode of excretion and degree or form of metabolism are some 15 of the targets for improved antibiotics.

Tylosin is a well known therapeutic agent in the veterinary field. (See, e.g., Tetrahedron Letters 1970, 2339, and U.S. Patent No. 3,178,341). Tylosin and tylosin-like macrolides have been modified in an attempt to obtain derivatives with improved properties. A large number of derivatives have been prepared, but improvement in activity has so far not been achieved to the desired degree.

It has now been found that reductive amination of the C-20 aldehyde group in tylosin and the aforementioned tylosin-like macrolides using certain cyclic amines as amination agents results in derivatives with significantly increased activity.

The macrolide derivatives prepared by the process of the present invention have the formula (I) 30 35

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/ \ -CHs 5 CH j Γ CH \ / \

| ^ Cha-Cha-R

Λ / CHa, / \ β γ \ (i)

1 o XOH Ϊ t / OH

L® Xrs 15 wherein R is a heterocyclic group containing a nitrogen atom as the sole heteroatom and bonded to the C₂Q carbonator through the nitrogen atom, the group being pyrrolidino, optionally with hydroxy, phenyl, piperidino or N, N-diethylcarbamoyl monosubstituted or methyl disubstituted piperidino , optionally trimethyl-substituted hexahydroazepino, 1,2,3,6-tetrahydropyridino, octahydroazocino, 1,2,3,4-tetrahydroquinolino or -isoquinolino, decahydroquinolino, octa-hydro-1H-azo-amino, azacyclotridecan 3-azabicyclo [3,2,2] nonane, 1-aza-spiro [4,5] decane or 1,3,3-trimethyl-6-azabicyclo [3,2,1] octane,

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Is hydrogen, hydroxy or mycarosyloxy

OH

30 T

• -i-CH

-0- # Y \ -0H

Ό- 'Y

OHs 35

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3 DK 153555B

R 2 is 9 Hs CH 3 / * ~ ° \. · —Α 5 H0- * * -0- or H0- * \ -o- ο6η3 X0CHs w / X ° CH3 4 Ί or acid addition salts thereof, provided that R if R is hydrogen, is (j! H3 15 H0-./ / * "° ~ cféhb xoch3 20 The analogous process of the present invention is characterized by (a) reducing an aldehyde of formula (III) in 25 i 0 cHy yv (iii) / cH3x / \ FT- ^ Hs-f 9 \

30 A / K Λ 1 ΐ OH

p * 7 VIV 0R v CH3 ^ # - N (CHs) 2

Cria XRs' 35

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wherein R 4 is R and R are as defined above and R is hydrogen or a hydroxy protecting group, in the presence of an amine of the formula HR, wherein R is as defined above, wherein any hydroxy protecting group R1 is removed to release the hydroxy group, or (b) reacting an amine of formula HR wherein R is as defined above with a macrolide of formula (IV) 10 \ \ *) \ (IV)

^ V-CH-CH-L

15 k A

4 / CH \ / \ R ^ H2-r T \ Λ · κ A, I oh 9H * N / iSV ^ i— <CHs or \ -N (CHa) e 20 wherein X 3a R, RJ and R4 are as defined above, and L is a leaving group which can be displaced by the amine HR in a non-reactive organic solvent, whereupon any hydroxy protecting group R is removed to release the hydroxy group and, if desired, converting a compound of formula (I) from reaction (a) or (b) to an acid addition salt thereof.

The term "hydroxy protecting group" refers to a substituent which is not removed under the reaction conditions but which can be easily removed after the reaction is completed to release the original hydroxyl group. hydroxy

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protecting groups are well known in the art (see e.g.

T.W. Greene, "Protective Groups in Organic Synthesis", Wiley-Interscience, 1981, pages 10-86). A particularly suitable hydroxy protecting group is the tetrahydropyranyl group.

5

Preparation of Macrolides of Formula (I)

Macrolides of formula (I) are prepared as mentioned from the corresponding macrolides of formula (II) in CH,

I I

CH₂ / jH-GHs-R5 (II) / CH \ / R R-CHs- /

Λ A Λ i I GH

CH2 \ / ^ \ / OR i- / CHa (CHa) 2

CtH3 20 1 3

wherein R is hydrogen or a hydroxy protecting group, R

And R is as defined above in formula (I) and R is -CHO or -C1 L. L represents a leaving group which can be displaced by an amine of formula HR wherein R is as defined in formula (I).

Thus, in accordance with one aspect of the present invention, macrolides of formula (I) are prepared by reductive amination of aldehydes of formula (II) wherein 5. 1 R is -CHO. Aldehyde starting materials wherein R is hydrogen include the following known macrolides of formula (II)

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Tylosin and its preparation by fermentation of Streptomyces fradiae NRRL 2702 or 2703 are described in U.S. Patent No. 3,178,341. Desmycosin and preparation of desmycosin by mild acid hydrolysis of tylosin are also described in U.S. Patent No. 3,178,341.

Macrocin and its preparation by fermentation of Streptomyces fradiae NRRL 2702 or 2703 are described in U.S. Patent 3,326,759. Lactenocin and preparation of lactenocin by mild acid hydrolysis of macrocine are also disclosed in U.S. Patent No. 3,326,759.

The aldehyde starting materials of formula (II) are converted to the amines of formula (I) by reduction in the presence of an amine of formula HR wherein R is as defined in formula (I). The preferred reducing agent is a cyanoborohydride of formula MBHgCN wherein M is a Group 1A or ammonium metal. Sodium cyanoborohydride is the reducing agent which is preferably selected. The reaction is preferably carried out using an excess amount of the amine of formula HR, typically from 2 to 3 equivalents. The solvent for the reaction is usually an inert polar solvent such as a C 1 -C 4 alkanol, preferably methanol. The reaction temperature may be from 0 to 60 ° C, preferably 20 to 40 ° C. Neutral conditions (pH 6-8) are preferred. Dehydrating agents, such as a 4A molecular sieve or anhydrous sodium or magnesium sulfate, may advantageously be used in the reaction.

Amines of formula (I) can also be prepared by reacting an amine of formula HR wherein R is as defined in formula (I), with a macrolide of formula (II) wherein R 30 is -CH 2 L and L is a leaving agent. group which can be displaced by the amine. Suitable leaving groups include e.g. trifluoromethanesulfonyl (triflate) and iodine.

C

Starting materials of formula (II) wherein R is -CH 2 L are conveniently prepared from the following known macrolides corresponding to formula (II) but where R 5 is -CH 2 OH: 8

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R3 R4 dihydrotylosin (relomycin) mycarosyloxy mycinosyloxy 5 dihydrodesmycosin OH mycinosyloxy CHa / * ~ ° \ dihydromacrocm mycarosyloxy HO- · ^ * -0— oti OCHa 10 9Hs / * “° \ dihydrolactenocin OH HO- · ^ / *

Clti ^ CHa 15 These macrolides are prepared by reducing the aldehyde group in tylosin, desmycosin, macrocin and lactenocin, respectively.

The C-20 hydroxyl group of the above macrolides is then converted to the desired leaving group L by 20 known methods. The C-20 hydroxyl group may e.g. is converted to the triflate group by reacting the macrolide with an activated derivative of trifluoromethanesulfonic acid, such as trifluoromethanesulfonic anhydride or trifluoromethanesulfonyl chloride, in the presence of a base in a non-reactive organic solvent. If desired, the triflate group can be converted to the iodine group, e.g. by reacting the uninsulated sulfone ester intermediate with a source of iodine idione such as tetra-n-butyl ammonium iodide or sodium iodide. In the case of dihydrodesmycosin and dihydrolactensin, the 20-iodine derivatives can be formed directly by adding iodine dissolved in a suitable solvent, such as dimethylformamide, to a solution of the 2

The leaving group at C-20 can then be exchanged by reaction with the amine HR in a suitable non-reactive organic solvent, such as acetonitrile, to give compounds of formula (I).

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The C-20 modified derivatives prepared according to the present invention form acid addition salts. These acid addition salts are also useful as antibiotics and are also prepared by the process of the present invention. Such salts are e.g. useful for separating and purifying the derivatives. In addition, the salts have an improved solubility in water.

Representative suitable salts include those salts formed by standard reactions with both organic and inorganic acids such as e.g. sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, cholic acid, pamoic acid, mucic acid, D-glutamic acid, D-camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, tartaric acid, tartaric acid stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, pyric acid, benzoic acid and cinnamic acid.

Illustrative C-20 modified derivatives prepared according to the present invention comprise the compounds listed in Tables I-IV.

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Table I

g

Illustrative C-20 Modified Derivatives of Tylosin

Compound No. _R_T6 octahydroazocin-1-yl T13 3-azabicyclo [3,2,2] nonan-3-yl

A

'O a r4 = HQ- · ° 9 r3 = mycarosyloxy π 6h3 chs

Table II

Illustrative C-20 modified derivatives of desmycosin 3

Compound No. «R_R

Dl pyrrolidin-1-yl OH

D2 piperidin-1-yl OH

D3 4-hydroxypiperidin-1-yl OH

D4 4-phenylpiperidin-1-yl OH

D5 hexahydroazepin-1-yl OH

D5a "H

D6 octahydroazocin-1-yl OH

D7 octahydro-1H-azonin-1-yl OH

DIO azacyclotridecan-1-yl OH

CH.3 30 / * ° \ a R4 = \ -0-, 5 i iH3 CHa 35

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Table II (continued) 3

Compound No. _R ^ _ R

Dll 1,2,3,4-tetrahydroquinolin-1-yl OH

D12 1 / 2,3,4-tetrahydroisoquinoline

5 -2-yl OH

D13 4-piperidinopiperidin-1-yl OH

Dl4 3-azabicyclo [3,2,2] nonan-3-yl OH

D15 3- (Ν, Ν-diethylcarbamoyl) -pipe-

ridin-1-yl OH

D19 1-azaspiro [4,5] decan-1-yl OH

10

D20 decahydroquinolin-1-yl OH

D21 1,3,3-trimethyl-6-azabicyclo-OH

[3.2.1] octane-6-yl

D22 1,2,3,6-tetrahydropyridin-1-yl OH

D23 3,3,5-trimethylbylhexahydroazepine OH

15 -1-yl (isomer 1)

D24 "(isomer 2) OH

D25 dodecahydrocarbazol-9-yl OH

D33 cis-3,5-dimethylpiperidin-1-yl OH

D34 trans-3,5-dimethylpiperidin-1-yl OH

20

Table III

Illustrative C-20 Modified Macrocin Derivatives

Compound No. _R_ M3 Hexahydroazepin-1-yl 25 M4 Octahydroazocin-1-yl (JJHa a 4 -3 30 R - HO- · ^ J and R = mycarosyloxy ηΠ QH3 35

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Table IV

Illustrative C-20 modified derivatives of lactenocina

Compound No. _R_ L4 hexahydroazepin-1-yl 5 L5 octahydroazocin-1-yl CH 2

, 0 and R4 = H0 - <^^> - 0-, R3 = OH

'ηΠ

Ana 15

The derivatives of the present invention inhibit the growth of pathogenic bacteria, especially gram positive bacteria, Mycoplasma species and gram negative bacteria such as Pasteurella species. The derivatives are particularly useful for Pasteurella species, such as P. multocida and P. hemolytica, and for Mycoplasma species, such as M. gallisepticum and M. hyopneumoniae (organism that causes mycoplasmapneumonia in pigs).

The minimum inhibitory concentrations (MICs) at which illustrative compounds inhibit certain bacteria are listed in Tables V and VI. The MIC of Table V is determined by standard agar dilution determinations. The MIC of Table VI is obtained using conventional nutrient thinning microtiter experiments.

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The C-20 modified derivatives prepared according to the present invention have shown in vivo antibacterial activity against laboratory-induced infections. If 2 doses of the test compound are administered 5 to mice experimentally infected with S.pyogenes C203, the observed activity is measured as an ED.

BU

tive dose in mg / kg to protect 50% of the test animals: see Warren Wick, et al., J. Bacteriol. 81, 233-235 (1961)). EDsQ values observed for illustrative compounds are listed in Table VII.

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Table VII

ED50 "Values of C-20 Modified Derivatives against Streptococcus pyogenes C203 in Mice _; __

Test compound '* 3 Subcutaneous Oral 5 - "" ---- -

Dl 1.2> 50 D2 0.9 50 D4 6.0 19 D5 1.3 50 10 D5a 1.5 34 D6 0.7 14 D7 1.6 12 D10> 10 68

Dll 7.5 19 D12 2.0 <6.3 15 D14 1.0 50 D19 2.9 46 D20 1.7 34 D21 1.0 10 20 D22 0.8 40 L5 1.8 100 M3> 10> 100 T6> 10 44 T13> 10 30 25 D33 0.625 7.98 D34 0.88 10.93

Desmycosin 1.03 84.1 mg / kg x 2, doses administered 1 and 4 hours post-infection 30 Compound number from Tables I-IV.

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Many of the C-20 modified derivatives prepared according to the present invention have shown in vivo anti-bacterial activity against infections caused by gram-negative bacteria. Tables VIII and IX summarize the results of experiments evaluating illustrative compounds against Pasteurella infections in one-day-old chicks. The compounds are administered parenterally or orally after infection of the chicks with Pasteurella multocida (0.1 ml -4 of a 10 dilution of a 24 hour old tryptose nutrient medium culture of poultry P. multocida administered subcutaneously). All untreated infected chickens die within 24 hours of Pasteurella infection in these trials.

In the experiments summarized in Table VIII, the compounds are administered by subcutaneous injection at a dose of 30 mg / kg 1 and 4 hours post-infection of the chicks with Ps multocida. In the experiments summarized in Table IX, the compounds are administered in treated drinking water (at a level of 0.53 g / liter) available from 4 to 20 hours prior to infecting the chickens with P. multocida and during the 3 day experimental 20 period.

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Table VIII

Activity of C-20 Modified Derivatives Subcutaneously Submitted to Pasteurella Multocida Infected Chicks 5 Experimental Number of Dead / Number of Treated

Dl 0/10 D2 0/10 D4 9/10 D5 0/10 10 D6 0/10 D7 3/10 D10 10/10

Dll 10/10 D12 9/10 15 D14 2/10 D19 0/10 D21 7/10 D22 0/10 D23 8/10 20 D24 2/10 D25 0/10 L5 Q / 10 25.

Subcutaneously administered, 30 mg / kg x 2.

30 Connection numbers from Tables II and IV.

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Table IX

Activity of C-20 Modified Derivatives Orally administered to Pasteurella multocida-infected chickens_ 5 Test compound0 Number of dead / number of treated

Dl 9/10 D2 5/10 D4 6/10 D5 2/10 10 D6 1/10 D7 2/10

Dll 8/10 D12 8/10 D14 0/10 15 D19 3/10 D20 0/10 D21 3/10 D22 5/10 D23 4/10 20 D25 7/10 D34 3/10, 0/10, 1/10 , 6/10

Desmvcosin 5/10 25 a

Administered in the available drinking water at a concentration of 0.53 g / liter.

3d

Connection number from Table II.

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The present compounds can be used to control infections caused by bacterial and Myco plasma species. In such a control, an effective amount of a compound of formula I is administered parenterally or orally to an infected or susceptible warm-blooded animal. The compounds may also be administered by insufflation, i.e. by blowing the compound in the form of a prepared powder into a closed area or room where the animals or poultry are kept. The animals or poultry breathe the pre-prepared powder present in the air. The prepared powder is also brought into the body through the eyes, a method known as intraocular injection.

The dose effective to control the infection varies with the severity of the infection and with the age, weight and condition of the animal. However, the total dose needed for parenteral protection is generally in the range of about 5%. 0.1 to approx. 100 mg / kg and preferably in the range of about 0.5 to approx. 50 mg / kg. The dose required for oral administration is generally in the range of 1 to approx. 300 mg / kg and preferably in the range of about 1 to approx. 100 mg / kg. Suitable dosage systems can be constructed.

The most convenient way of administering the compounds is often by formulation in the feed supply or in the drinking water. Various feedstuffs, e.g. ordinary dry feed, liquid feed and pellet feed can be used.

Furthermore, the compounds of the present invention may be included in compositions useful for controlling infections caused by bacterial and Mycoplasin species. These compositions 30 contain a compound of formula (I) together with a matrix. Preparations may be formulated for parenteral or oral administration by methods recognized in the pharmaceutical art.

The methods for formulating pharmaceutical substance in animal feed are well known. A preferred method is to prepare 3-

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a premix of concentrated pharmaceutical which is again used in the preparation of prepared feed.

Typical premixtures may contain from ca. 1 to approx. 200 g 5 pharmaceutical substance per kg of premix. Premixtures can be either liquid or solid formulations.

The final formulation of animal or poultry feed depends on the amount of pharmaceutical to be administered. The usual methods of formulation, mixing and pelleting can be used to prepare feed containing a compound of formula (I).

Effective injectable compositions containing these compounds may be in the form of either suspension or solution. In the preparation of formulations, it will be known that the water solubility of the acid addition salts is generally greater than that of the free bases. Similarly, the bases are more soluble in dilute acids or in acidic solutions than in neutral or basic solutions.

In the solution form, the compound is dissolved in a physiologically acceptable matrix. Such matrixes contain a suitable solvent, if necessary preservatives such as benzyl alcohol, and buffers. Useful solvents include e.g. water and aqueous alcohols, glycols and carbonates such as diethyl carbonate. Such aqueous solutions generally do not contain more than 50% by volume of the organic solvent.

Injectable suspension preparations require a liquid suspending medium, with or without excipients, as a matrix. The suspension medium may e.g. be aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or highly refined mineral oils, or aqueous carboxymethyl cellulose.

Suitable physiologically acceptable excipients are needed to keep the compound suspended in the suspension preparations. The excipients can be selected from the thickening agent.

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agents such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin and the alginates. Many surfactants are also useful as suspending agents. Lecithin, alkylphenol polyethylene oxide addition products, naphthalene sulfonates, alkylbenzenesulfonates and polyoxyethylene sorbitan esters are useful suspending agents.

Many substances that affect the hydrophilicity, density and surface tension of the liquid suspending medium can assist in the preparation of injectable 10 suspensions in individual cases. Eg. For example, silicone antifoams, sorbitol and sugars may be useful suspending agents.

For further illustration of the present invention, the following examples are provided. In these examples, the abbreviation "20-DH-DO" is used for the term "20-dihydro-20-deoxy".

Preparation 1 20-Dihydrotylosin (relomycin) 20 30.0 g (32.8 mmol) of a solution of tylosin base in 300 ml of 2-propanol and 200 ml of water are treated portionwise with 315 mg (8.2 mmol) of sodium borohydride over 5 minutes. 30 minutes after the addition is complete, the pH of the reaction solution is adjusted to 7.0 by the addition of 1N sulfuric acid solution. The neutralized solution is evaporated in vacuo to remove 2-propanol, the remaining aqueous solution is treated with 500 ml of a saturated sodium bicarbonate solution. The mixture is extracted 3 times with dichloromethane, each time with 300 ml, and the combined extracts are extracted with 200 ml of a saturated sodium chloride solution and dried over sodium sulfate. Filtration followed by evaporation yields a glass which is broken up into n-hexane, collected on a filter and air dried to give 28.5 g (95%) of 20-dihydrochylosin.

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Preparation 2 20-Dihydrodesmycosin 10 g (13 mmol) of desmycosin, dissolved in isopropanol: water (1: 1, 175 ml), is stirred at room temperature while 125 mg of 5 (3.3 mmol) of NaBH4 is added. After 1/2 hour, the pH of the reaction mixture is adjusted to 7.0 with 1 N 1 SO 2. The alcohol is removed under reduced pressure. A saturated NaHCO 3 solution is added to the aqueous solution and the product is extracted into CH 2 Cl 2 · The organic layer is dried (Na 2 SO 4) and the solvent is removed under reduced pressure to give 9.65 g (12.5 mmol, 96%). yield) 20-dihydrodesmycosin as a white foam.

Preparation 3 20-DH-DQ-20-Iodine Desmycosin (Method 1) Dissolve 2.0 g (2.6 mmol) of 20-dihydrodesmycosin and 1.5 g (3.9 mmol) of tetra-n-butylammonium iodide in 30 ml of CH 2 Cl 2 to which is added 0.6 ml (4.5 mmol) of s-collidine. This solution is cooled to -78 ° C under a nitrogen atmosphere and treated dropwise with a syringe with 0.6 ml (3.9 mmol) of trifluoromethanesulfonic anhydride. The reaction is stirred for 5 minutes at -78 ° C and then allowed to come to room temperature (approx.

30 minutes). A saturated NaHCO 3 solution is added and the product is extracted with CH 2 Cl 2 - The organic layer is dried (Na 2 SO 4) and evaporated to give a red oil which is purified by silica gel line chromatography eluting initially with 400 ml of CH 2 Cl 2 and then stepwise with CH 2 Cl 2: CH 2 OH solutions as follows: 98: 2 (250 ml), 96: 4 (500 ml), 95: 5 (250 ml}, 94: 6 (750 ml) 30 and 92: 8 (250 ml). desired product is identified by TLC, combined and evaporated to dryness to give 595 mg (0.67 mmol, 26% yield) of 20-DH-DO-20-iodine desmycosin as a white foam.

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Preparation 4 2Q-DH-DQ-2Q-Iodesmycosin (Method 2).

5/0 g (6/5 mmol) of 20-dihydrodesmycosin and 2.54 g (9.70 mmol) of triphenylphosphine are dissolved in 10 ml of di-ethylformanide (ETC). This mixture is stirred at room temperature under N 2, while 2 g of 46 g (9.70 mmol) of iodine in 5 ml of DMF is added dropwise. The reaction mixture is stirred for 2 hours bg then poured into a cold saturated

NaHCO solution. The product is extracted twice with CHCl 3 and the combined CHCl 3 extracts are shaken with 0.1 mol of 10 ύ sodium thiosulfate to remove unreacted iodine. The organic layer is dried (Na 2 O 4) and evaporated under reduced pressure to give a light yellow oil which is purified by silica gel line chromatography. The column is initially eluted with 500 ml CH2 Cl2 and then with 250 ml portions of CH2 Cl2 RCH2 OH-15 mixtures as follows: 98: 2, 96: 4, 95: 5, 94: 6, 92: 8, 88:12 and 86 : 14th Fractions containing the desired product are identified as in Preparation 3 and combined to give 1.78 g (2.0 mmol, 31% yield) of 20-DH-DO-20-iodine desmycosin as a white foam.

20

Example 1 20-DH-DO-2Q- (Octahydroazocin-1-yl) -desmycosin 575 mg (0.65 mmol) of 20-DH-DO-20-iodine desmycosin is dissolved in 10 ml of acetonitrile and 0.37 g (0, 41 ml, 3.3 mmol) of heptamethylene imine are added to this solution. The reaction is stirred at reflux for 1.5 hours. Then, volatiles are removed under vacuum. The residue is dissolved in CH 2 Cl 2 and extracted with a saturated NaHCO 3 solution. The organic layer is dried (Na 2 SO 4) and then evaporated under reduced pressure to give a light brown foam. This foam is purified by silica gel line chromatography eluting with 250 ml of each of the following CH 2 Cl 2: CH 3 OH mixtures: 98: 2, 96: 4, 94: 6, 9: 1, 88:12, 82:18, 65:35, 1: 1, 1: 3 and finally with 300 ml of CH 2 OH.

Fractions containing the desired product are identified by TLC, combined and evaporated to dryness to give 397 mg (0.46 mmol, 71% yield) of 20-DH-DO-20- (octahydroazocin-1-yl) -desmycosin as a white foam.

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Example 2 20-DH-DO-2Q- (Hexahydroazepin-1-yl) -desmycosin 10 g (13 mmol) of desmycosine dissolved in 100 ml of anhydrous methanol is rapidly added to a solution of 3.3 g (52 mmol) of 5 NaBH and 6.5 g (7.5 ml, 65 mmol) of hexamethylenimine in 50 ml of anhydrous methanol underneath. The reaction mixture is stirred under N 2 at room temperature for approx. 3 hours and then evaporated under reduced pressure. The resulting residue is dissolved in just enough ethyl acetate to aid in dissolving the residue, and this solution is extracted with a saturated NaHCO 3 solution. The organic layer is separated, dried (Na 2 SO 4) and evaporated under reduced pressure to give a pale yellow foam. This foam is purified by silica gel line chromatography, initially eluting with 1 liter of CH 2 Cl 2, and then incrementally with 500 ml portions of CH 2 Cl 2 CH 2 OH mixtures as follows: 98: 2, 96: 4, 94: 6, 92: 8 and 9 : 1, and finally with CH 2 Cl 2: CH 3 OH: NH 4 OH mixtures as follows: 90: 10: 0.5 (500 ml) and 75: 25: 0.5 (2 liters).

ΡΩ

Fractions containing the desired product are identified by TLC, combined and evaporated to dryness to give 6.035 mg (7.07 mmol) of 20-DH-DO-20- (hexahydroazepin-1-yl) desmycosin as a white foam. Other fractions containing impure product 3 are combined, redissolved in CH 2 Cl 2, extracted again with a saturated NaHCO 3 solution and purified as above using a silica gel column filled with CH 2 Cl 2: CH 3 OH (9: 1) eluting with CH 2 Cl 2: CH 3 OH: NH 4 OH as follows: 90: 10: 0.5 (500 ml) and 80: 20: 0.5 (1 liter) to give an additional 1.372 g (1.61 mmol) of product.

The total yield of 20-DH-DO-20- (hexahydroazepin-1-yl) desmycosin is 7.407 g (8.68 mmol, 67% yield).

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Example 3 20-DH-DO-2Q- (4-Phenylpiperidin-1-yl) -desittycosine 1.5 g (2 mmol) of desmycosine is dissolved in 60 ml of absolute methanol and treated with 640 mg (4 mmol) of 4-phenylpiperidine in the presence of "Linde ^ A" molecular sieves. After 1/2 hour, 500 mg (8 mmol) of NaBH 3 CN is added and the mixture is stirred for 2.5 hours at room temperature. The mixture is poured into 200 ml of a saturated NaHCO 3 solution and extracted 3 times with Cl 2 C 2, each time with 200 ml. The combined organic extracts are dried (Na 2 SO 4), filtered and evaporated under reduced pressure. The 3.6 g residue is purified by flash chromatography on silica gel eluting with a gradient of 1 liter of CH 2 Cl 2 to 1 liter of MeOHsCE 2 C 2 (5:95) and then with 1 liter

MeOH: CH ~ Cl ~ (5:95). Fractions containing the desired product are located by TLC, combined and evaporated to dryness to give 680 mg of 20-DH-DO-20- (4-phenylpiperidin-1-yl) desmycosin.

Example 4 20-DH-DO-20- (Hexahydroazepin-1-yl) -41-deoxydesmycosin

A solution of 565 mg (0.75 mmol) of 4'-deoxydesmycosin in 15 ml of methanol under argon is stirred with 2.2 g of activated "Lind ^ B) 3A" molecular sieves for 30 minutes ^ before 0.25 ml (2.25 mmol 25 hexamethylenimine is added. 1 hour later, 141 mg (2.25 mmol) of sodium cyanoborohydride is added to the solution. After a further 45 minutes, the reaction mixture is poured into a saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic extracts are shaken with a saturated sodium chloride solution, dried over sodium sulfate, filtered and evaporated to give 600 mg of crude product.

This product is purified by preparative TLC on silica gel eluting with dichloromethane: methanol: concentrated ammonium hydroxide (90: 15: 2) to give 150 mg (24% yield) of 20-DH-DO-20- (hexahydrozepin-1-yl) -4'-deoxydesmycosin.

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Examples 5-6 20-DH-DO-20- (Octahydroazocin-1-yl) -desmycosin are prepared analogously to the method of Example 2.

20-DH-DO-20- (Hexahydroazepin-1-yl) -desmycosin

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is made analogous to the method of Example 1.

Example 7 20-DH-DO-2Q- (Octahydroazocin-1-yl) -desmycosin (Method 3) 1Q 4.0 g (5.2 mmol) of desmycosin is dissolved in 30 ml of absolute methanol and treated with 1.2 g (1 , 3 ml, 10.4 mmol) of heptamethylenimine in the presence of 3A molecular sieves. After the reaction mixture has been stirred for 1 hour at room temperature, a solution of 60 mg (1.6 mmol) of NaBH 3 in 10 ml of absolute methanol is rapidly added by means of a pipette.

The reaction mixture is stirred for 1.5 hours at room temperature and then an additional 30 ml of NaBH 3 (in one portion as solid) is added. The reaction mixture is stirred for a further 75 minutes and then filtered. The filtrate is evaporated under reduced pressure. The residue is dissolved in 150 ml of ethyl acetate and this solution is extracted with 150 ml of water and 100 ml of a saturated NaHCO 3 solution. Then the ethyl acetate solution is extracted with 150 ml of a 0.5 M NaE 2 PO 2 buffer of pH 6.5. The buffer extract is evaporated under vacuum 25 to remove residual ethyl acetate and stirred rapidly while 5N NaOH is added slowly to give a thick white precipitate. The white solid is removed by filtration, washed with a small amount of water and dried to give 3.55 g of 20-DH-DO-20- (octahydroazocin-1-yl) -3Q-desmycosin.

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Example 8 20-DH-DO-2Q- [1-Azaspiro [4,51decan-1-yl] -desmycosin 5/0 g (6.5 mmol) of desmycosin is dissolved in 50 ml of absolute methanol and treated with 1.36 g ( 9.8 mmol) l-azaspiro [4,5] -5 decane in the presence of 3A molecular sieves. After 15 minutes, 620 mg (9.8 mmol) of NaBH 3 CN is added and the mixture is stirred for 17 hours at room temperature. The reaction mixture is filtered and the filtrate is evaporated under reduced pressure. The residue is dissolved in 300 ml of ethyl acetate and extracted with 300 and 100 ml of water. Then, the product from the ethyl acetate solution is extracted with 300 ml and 100 ml of a 0.5 M NaH + TC 2 buffer of pH 6.5. The phosphate buffer extracts are combined and evaporated in vacuo to remove residual ethyl acetate. The phosphate buffer solution is then stirred rapidly while 5 N 15

NaOH is added slowly, giving a thick white precipitate. The white solid is removed by filtration, washed with water and dried to give 3.52 g of 20-DH-DO-20 - [1-azaspiro [4,5] decan-1-yl] -desmycosin.

20

Example 9 20-DH-DO-2Q- (1,2,3,4-Tetrahydroquinolin-1-yl) -desmycosin 11.6 g (15 mmol) of desmycosine is dissolved in 100 ml of dry methanol and 3.8 ml (30 ml). mmol) 1,2,3,4-tetrahydroguinoline is added. After the mixture is stirred for 30 minutes at room temperature, 1.25 g (20 mmol) of sodium cyanoborohydride are added. The mixture is stirred overnight and then evaporated under reduced pressure. The residue is partitioned between 100 ml of ethyl acetate and 100 ml of water. Thereafter, the organic layer is extracted in that order with 100 ml of an aqueous pH 30 phosphate buffer 6.5 and 100 ml of an aqueous pH phosphate buffer 4.5. The ethyl acetate layer is dried over sodium sulfate, filtered and evaporated and the residue of 4.6 g is separated by chromatography on silica gel ("Waters Prep 500"). The column is eluted with a linear gradient of 4 liters of OO i

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dichloromethane and 5% methanol plus 0.5% concentrated ammonium hydroxide in 4 liters of dichloromethane. Fractions containing the desired product are identified by TLC analysis, collected and evaporated to dryness to give 3.4 g of the title compound ice.

Example 10 20-DH-DO-2Q- (1,2,3,4-Tetrahydroisoquinolin-2-yl) -desmycosin 11.6 g (15 mmol) of desmycosin is dissolved in 100 ml of dry methanol and 3.8 ml (30 mmol) 1 , 2,3,4-tetrahydroisoquinoline is added. After the mixture has been stirred for 30 minutes at room temperature, 1.25 g (20 mmol) of sodium cyanoborohydride are added. The mixture is stirred overnight and then evaporated under reduced pressure. The residue is partitioned between 150 ml of ethyl acetate and 150 ml of water. Then, organic layers are extracted in the order of 100 ml of phosphate buffer pH 6.5 and 100 ml phosphate buffer pH 4.5. After evaporation of the buffer extract of pH 4.5 under reduced pressure to remove ethyl acetate, the pH of 10 is adjusted with 5 N sodium hydroxide. The precipitate formed is collected and air dried to give 5.6 g of the title compound.

Example 11 20-DH-DO-2Q- (1,2,3,6-Tetrahydropyridin-1-yl) -desmycosine 11.6 g (15 mmol) of desmycosine is dissolved in 100 ml of dry methanol and 2.8 ml (30 mmol) 1,2,3,6-tetrahydropyridine is added. After the mixture is stirred for 30 minutes at room temperature, 1.25 g (20 mmol) of sodium cyanoborohydride are added. The mixture is stirred overnight and then evaporated under reduced pressure. The residue is dissolved in 150 ml of ethyl acetate. This solution is extracted with 150 ml of water and then twice with an aqueous phosphate buffer solution of pH 6.5, each time with 100 ml. The buffer solutions 35 are evaporated separately under reduced pressure to remove

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ethyl acetate and then adjusted to pH 10 with 5 N

sodium hydroxide. The precipitates formed are collected by filtration and air dried to give 5.4 g (first extract) and 3.2 g (second extract) of the title compound.

5

Examples 12-29

The following examples are prepared analogously to the methods described in Examples 1, 2, 7 or 8: 20-DH-DO-20- (octahydroazocin-1-yl) -lactenocin, 20-DH-DO-20 (pyrrolidin-1-yl) ) -desmycosin, 20-DH-DO-20- (azacyclotridecan-1-yl) -desmycosin, 20-DH-DO-20- (4-hydroxypiperidin-1-yl) -desmycosin, 20-DH-DO-20 (hexahydroazepin-1-yl) macrocin., 20-DH-DO-20- [3-azabicyclo [3,2,2] -nonan-3-yl-desmycosin, 20-DH-DO-20- (piperidine -1-yl) -desmycosin, 20-DH-DO-20- [3-. (N, N-Diethylcarbamoyl) -piperidin-1-yl] desmycosin, 20-DH-DO-20 - [(4-piperidino) -piperidin-1-yl] -desmycosin, 20-DH-DO-20 (octahydro-1H-azonin-1-yl) -desmycosin, 20-DH-DO-20- (decahydroquinolin-1-yl) -desmycosin, 20-DH-DO-20- [1,3,3-trimethyl] 6-azabicyclo [3,2,1] octan-6-yl] -desmycosin, 20-DH-DO-20- (dodecahydrocarbazol-9-yl) -desmycosin, 20-DH-DO-20- (octahydroazocin) 1-yl) -ylosin, 20-DH-DO-20- (3-azabicyclo [3,2,2] nonan-3-yl) -ylosin, 20-DH-DO-20- (octahydroazocin-1-yl) ) -macrocin, 20-DH-DO-20- (hexahydroazepin-1-yl) -lactenocin.

30 35

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38 DK 153555 B

Examples 30-33 20-DH-DO-20- (3,3,5-Trimethylhexahydroazepin-1-yl) - desmycosin are prepared analogously to the method of Example 85 and then separated into individual isomers 1 and 2 by silica gel line chromatography.

20-DH-DO-20- (Dodecahydrocarbazol-9-yl) -desmycosin (compound D25) is a mixture of 2 isomers. The mixture is separated into 2 fractions, each rich in one of the isomers, 1Q by silica gel line chromatography. Each of the isomer-enriched fractions has an activity pattern that is similar to that of the mixture.

EXAMPLES 34-35 15 The 20-DH-DO-20- (Octahydroazocin-1-yl) -desmycosin dihydrochloride and tartrate are prepared from the 20-DH-DO-20- (octahydroazocin-1-yl) -desmycosin using standard methods.

2Q Table X-XIII summarizes certain physical data for the exemplified compounds.

25 30 35

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Claims (3)

44 DK 153555B O Patent claims. An analogous process for the preparation of 20-amino derivatives of tylosin, desmycosin, macrocine or lac-tenocin of formula (I) in io / p * - CHa / * * in 23 CHS / R4-CH2-j / \ Wherein CH is a heterocyclic group containing a nitrogen atom as the sole heteroatom and bonded to 15 CHs ^ \ / j [\ / Nh Ϊ_ / 0H I cr 0 · / "TV CHs CQ VN (CHa) a \ _ ± / L Ns CHa 20 The C2C carbonator through the nitrogen atom, the group being pyrrolidino, optionally with hydroxy, phenyl, piperidino or N, N-diethylcarbamoyl monosubstituted or with methyl disubstituted piperidino, optionally trimethylsubstituted hexahydroazepino, 1,2,3,6-tetrahydropyridino, 2,3,4-tetrahydroquinolino or isoquinolino, decahydroquinolino, octahydro-1H-azonino, azacyclotridecane, dodecahydrocarbazole, 3-azabicyclo [3,2,2] nonane, 1-aza-spiro [4,5 ] decane or 1,3,3-trimethyl-6-azabicyclo [3.2.1] octane, R is hydrogen, hydroxy or mycarosyloxy 45 DK 153555B Γ, · - · ς -— CH3 -O- ·. · —OH 5 χ CH 3 4 R is 10 CHs CHa a- * χ / * - 0- or HO- * '^ * - 0- 0 ^ h3 OCH3 HC that R if R is hydrogen is 20 CHs κ * ~ \ HO - \ - 0- \ -fi 0ΌΗ3 X0CH3 25 characterized in that (a) reduces an aldehyde of formula (III) 30 \ 35 O 46 DK 153555B i * '' V-CHa 5. II o CHy yJH ^ Hy * CHy \ <iH> 10 cH / V / fX. hydroxy protecting group, in the presence of an amine of formula HR, wherein R is as defined above, then any hydroxy protecting group R3 "is removed to release the hydroxy group, or (b) reacting an amine of formula HR where R is as defined above , with a macrolide of formula (IV) 25 | CHa / \ (IV) Y CH 2 -CH 2 —L 30, / CH \ y \ R '^ I \ ch / \ / | \ / xor1 ^^ - N (CHs) e 35 Ciis \ z O. In which 1 to 4 R, RJ and R are as defined above and L is a leaving group which can be displaced by the amine HR in a non-reactive organic solvent, whereupon an optional hydroxy in protecting group R is removed to release the hydroxy group and, if desired, converts a compound of formula (I) from reaction (a) or (b) into an acid addition salt thereof. An analogous process according to claim 1 for the preparation of a macrolide of formula (I) or a pharmaceutically acceptable salt thereof, characterized in that R 1 is mycinosyloxy, and R 2 is hydroxy. The analogous method of claim 1 for the preparation of 20-dihydro-20-deoxy-20- [3-azabicyclo [3,2,2] nonan-3-yl] -desmycosin. 20 25 30 35